Epstein-Barr virus (EBV) is associated with a variety of human cancers including Hodgkin's lymphoma, primary central nervous system lymphoma in AIDS, systemic lymphoma in AIDS, post-transplant lymphoproliferative disease (PTLD), nasal T cell lymphoma, undifferentiated gastric carcinoma, and nasopharyngeal carcinoma (Filipovich et al., In I. T. Magrath (ed.), The Non-Hodgkin's Lymphomas, (Williams and Wilkins, 135-154 (1990)); Herbst et al., Proc. Natl. Acad. Sci., 88,4766-4770 (1991); MacMahon et al., Lancet, 338, 969-973 (1991); Miller, In B. N. Fields et al. (eds.), Virology, 2nd ed. (Raven Press, New York, 1921-1958, 1990); Staal et al., Am. J. Clin. Pathol., 91, 1-5 (1989)). There also has been a report of an association between EBV and human breast cancer. Primary infection in young adults results in infectious mononucleosis. Once infected, the individual carries the virus in B cells as a latent infection for life. Approximately 80-90% of the adult population in the United States is infected with this virus. In most cases the virus and host coexist uneventfully. However, the onset of immunosuppression, either clinically-induced in the case of transplant patients, or present as a consequence of other infections, for example in AIDS, leads to an increased risk for the development of EBV-associated malignant disease. There currently is no treatment available to eliminate the B cells latently infected with EBV that are the progenitors of these malignancies. Nucleoside analogs have some efficicacy in reducing lytic EBV infection but have no effect on the latently replicating virus since the EBV DNA polymerase and EBV thymidine kinase enzymes, which are the targets of the nucleoside analogs, are not expressed during latent infection. Accordingly, there remains a need for methods to prevent de novo EBV infection, and to treat latent EBV infection, particularly following organ transplantation, given that EBV plays such a substantial role in postransplantation morbidity and mortality (Kumar et al., Am. J. Surg. Pathol., 17, 1046-1053 (1993); Randhawa et al., Hepatology, 21, 1751 (1995); Rosendale et al., Arch. Pathol. Lab. Med., 119, 418-423 (1995)).
The EBV EBNA2 protein is one of the first viral proteins expressed after infection by EBV. EBNA2 is a transcriptional activator that regulates viral latency gene expression and activates expression of cellular genes (Abbott et al., J. Virol., 64, 2126-2134 (1990); Calender et al., Proc. Natl. Acad. Sci., 84, 8060-8064 (1987); Cohen et al., J. Virol., 65, 5880-5885 (1991); Cordier et al., J. Virol., 64, 1002-1013 (1990); Ling et al., Proc. Natl. Acad. Sci., 90, 9237-9241 (1993a); Rickinson et al., J. Virol., 61, 1310-1317 (1987); Rooney et al., J. Virol., 66, 496-504 (1992); Wang et al., J. Virol., 64, 2309-2318 (1990)). EBNA2 is critical for the establishment of a latent infection in the B cell and for initiating the changes in B cell growth that can ultimately lead to tumorigenesis. On primary infection of B cells, the latency W promoter (Wp) is used to express EBNA-LP and EBNA2 (Sample et al., Proc. Natl. Acad. Sci., 83, 5096-5100 (1986); Speck et al., Proc. Natl. Acad. Sci., 82, 8305-8309 (1985)). Expression of the EBNA genes then switches from the Wp to the latency C promoter (Cp), and this switch is controlled by EBNA2 (Bodescot et al., J. Virol., 61, 3424-3430 (1987); Rooney et al., J. Virol., 63, 1531-1539 (1989); Rooney et al. (1992), supra; Woisetschlager et al., Proc. Natl. Acad. Sci., 87, 1725-1729 (1990); Woisetschlager et al., Proc. Natl. Acad. Sci., 88, 3942-3946 (1991)). The promoters for the latency membrane proteins LMP-1 and LMP-2 (terminal protein) are also up-regulated by EBNA2 (Fahraeus et al., Proc. Natl. Acad. Sci., 87, 7390-7394 (1990); Tsang et al., J. Virol., 65, 6765-6771 (1991); Wang et al., J. Virol., 65, 4101-4106 (1991); Zimber-Strobl et al., J. Virol., 65, 415-423 (1991); Zimber-Strobl et al., EMBO J., 12, 167-175 (1993)), placing the entire program of latency gene expression under the influence of EBNA2. Further, the changes in surface expression of B cell activation antigens that are induced by EBV infection (Calender et al., Proc. Natl. Acad. Sci., 84, 8060-8064 (1987); Rowe et al., EMBO J., 6, 2743-2751 (1987)) are also recognized as being partially attributable to EBNA2. In particular, expression of CD21 and CD23 has been shown to be upregulated by EBNA2 (Cordier et al., supra; Rowe et al., supra; Wang et al., Proc. Natl. Acad. Sci., 84, 3452-3456 (1987); Wang et al. (1991), supra). Activation of cellular genes by EBNA2 thus appears to have an important role in altering B cell growth control.
The mechanism of EBNA2-mediated transactivation has become an area of intense investigation. EBNA2 does not bind directly to DNA, but rather targets promoters through interaction with a cellular DNA-binding protein designated CBF1 that binds to genes having upstream CBF1 binding sites (Ling et al., J. Virol., 68, 5375-5383 (1994); Ling et al., J. Virol., 67, 2990-3003 (1993b); Zimber-Strobl et al. (1993), supra). Peptide sequencing and cloning recently revealed CBF1 to be identical to recombination binding protein J kappa (RBPJk) (Grossman et al., Proc. Natl. Acad. Sci., 91, 7568-7572 (1994); Henkel et al., Science, 265, 92-95 (1994)). This latter protein was named on the basis of its ability to bind to the heptamer sequence in the immunoglobulin J kappa gene (Matsunami et al., Nature, 342, 934-937 (1989)). However, this binding ability was subsequently found to be artifactually generated by the addition of a BamHI linker to the heptamer probe (Grossman et al., supra; Henkel et al., supra). CBF1/RBPJk is highly conserved in sequence between species as divergent as humans and members of the genus Drosophila (Amakawa et al., Genomics, 17, 306-315 (1993); Furukawa et al., J. Biol. Chem., 266, 23334-23340 (1991); Schweisguth et al., Cell, 69, 1199-1212 (1992)). In particular, the Drosophila homologue is encoded by the suppressor of hairless gene, and plays a key role in determination of neuronal cell fate (Furukawa et al., supra; Schweisguth et al., supra).
An examination of the binding site for CBF1/RBPJk reveals an essential core sequence, GTGGGAA that is necessary for binding, with flanking sequences influencing binding affinity (Ling et al. (1994), supra). The acceptable flanking sequences further have been defined by binding-site selection (Tun et al., Nucleic Acids Res., 22, 965-971 (1994)), and a database search using this consensus sequence identifies CBF1/RBPJk-binding sites in a large number of cellular promoters. This confirms that EBNA2 has substantial potential to reprogram B cell gene expression. Along these lines, CBF1/RBPJk acts as a transcriptional repressor and may be a significant contributor to the downregulation of genes such as the surface activation antigens that are silent in quiescent B cells. By targeting CBF1/RBPJk, EBNA2 short-circuits this aspect of B cell regulatory control and can activate the CBF1/RBPJk repressed genes in the absence of the normal B cell proliferation signals.
In an effort to better understand the EBNA2 protein, the EBNA2 gene of the baboon lymphotropic virus, Herpesvirus papio (HVP), has been cloned and sequenced (Ling et al. (1993b), supra). A comparison of its amino acid sequence with that of the human type A (e.g., strain B95-8 or W91) and human type B (e.g., strain AG876) EBNA2 proteins (Dambaugh et al., Proc. Natl. Acad. Sci., 81, 7632-7636 (1984)) reveals nine conserved regions, i.e., CR1 through CR9. CR8 5 contains the critical hydrophobic segment of the activation domain, and CR9 is a strong karyophilic signal sequence (Cohen et al. (1991), supra; Cohen et al., J. Virol., 65, 2545-2554 (1991a); Ling et al. (1993b), supra). The conserved regions CR5, CR6, and CR7, which encompass the amino acids 252-425, contain the CBF1/RBPJk interaction domain in EBNA2.
A role for CR6 in CBF1/RBPJk binding previously had been suggested by the inability of a peptide carrying a double mutation of tryptophans 323 and 324 to interact with CBF1/RBPJk in an electrophoretic mobility shift assay (EMSA) (Ling et al. (1993a), supra; Ling et al. (1994), supra). However, these studies did not elucidate further critical regions of the protein necessary for CBF1 interaction. Similarly, through analysis of glutathione S-transferase (GST)-EBNA2 fusion proteins, EBNA2 amino acids 310-336 were identified as sufficient for CBF1/RBPJk interaction, and either the shorter sequence PPWWPP (i.e., Pro Pro Trp Trp Pro Pro [SEQ ID NO: 1]) or the longer sequence GPPWWPP (I/V) (C/R) DP (i.e., Gly Pro Pro Trp Trp Pro Pro (Ile/Val) (Cys/Arg) Asp Pro [SEQ ID NO: 2]) was suggested as possibly mediating this interaction (Tong et al., J. Virol., 68, 6188-6197 (1994); Grossman et al., supra; Yalamanchili et al., Virology, 204, 634-641 (1994)). However, none of these studies went so far as to actually characterize the functional region for interaction, for instance, by determining whether a peptide comprising the region can compete with the native (i.e., wild-type) EBNA2 protein for CBF1 interaction. Furthermore, none of these studies has resulted in the synthesis of an EBNA2 peptide having sufficient biological activity (i.e., ability to compete with the native (i.e., wild-type) EBNA2 protein for CBF1 interaction) so as to comprise a potentially therapeutically effective clinical pharmaceutical agent.
Thus, precise definition of the CBF1/RBPJk interaction domain in EBNA2 would allow peptides comprising this region to be synthesized. This offers the possibility of manipulation of viral and/or cellular gene expression through application of the peptides. For instance, such peptides would find use in the study of B cell differentiation and modification thereof by EBV infection. Moreover, such peptides could be employed, for instance, in disrupting the interaction between CBF1 and EBNA2. This would allow the therapeutic use of the peptides, particularly as an anti-EBV agent (e.g., in preventing de novo EBV infection) and as an antitumor agent (e.g., in preventing EBNA2-initiated changes in B cell growth that can ultimately lead to tumorigenesis).
Accordingly, the present invention seeks to overcome at least some of the aforesaid limitations of the prior art. In particular, it is an object of the present invention to provide EBNA2 peptides comprising the CBF1/RBPJk interaction domain. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.